Cholesterol control agent

ABSTRACT

The invention relates to medicine and can be used for producing a medicinal agent for normalising the cholesterol level. The aim of the invention is to develop a physiologically acceptable cholesterol-control agent which is nontoxic and does not cause addiction of organism. In preferred embodiments, the method for producing said agent consists in using 2-6 weight parts of a 36.5-40.0% formaldehyde medicinal solution and in adding 998-994 weight parts of a sterile 0.85-0.95% sodium chloride solution which is used for injection in such a way as to produce a 0.07-024% formaldehyde solution. The agent is stored at a temperature of 15-35° C. It also encompasses a method for treatment of patients for lowering their cholesterol levels that comprises administering the agent to the patients as intramuscular injections with a predetermined dose at a predetermined time interval.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a U.S. national phase application of a PCTapplication PCT/RU2008/000203 filed on 2 Apr. 2008, published asWO2009/005396, whose disclosure is incorporated herein in its entiretyby reference, which PCT application claims priority of a Russian patentapplication RU2007/125298 filed on 4 Jul. 2007.

FIELD OF THE INVENTION

The invention is related to medicine and can be used to make acholesterol-lowering drug.

BACKGROUND OF THE INVENTION

Blood cholesterol is an important characteristic of lipid metabolism.Cholesterol-lowering therapy provided for patients with atherosclerosisand coronary heart disease results in a reduction of the cardiovasculardeath rate by 30-40%. There is a direct relationship between bloodcholesterol levels and coronary diseases.

Cholesterol-lowering drugs are known which are based on plantpreparations. In particular, a cholesterol-lowering drug (see RF PatentNo. 2162333 according to IPC A61K35/78, published on Jan. 27, 2001) isknown, which contains Licopodium (wolfs-claws), Aurum iodatum (auriciodate), Barium carbonicum (barium carbonate), Alluim sativum (garlic)taken in equal proportions at CI2 dilution. This drug is a mix ofhomeopathic preparations, each of these preparations being used forspecific indications from arterial hypertension and atheroscleroticprocesses to chronic liver disease, urolithiasis, adiposity, etc. Use ofa multicomponent drug like that described above is equivalent topolyprogmasy which usually makes patient's response to treatmentdifficult to interpret and ultimately precludes from making up anoptimal treatment plan.

Another cholesterol-lowering drug based on plant preparation (see RFPatent No. 2152221 according to IPC A61K35/78, published on Jul. 10,2000) is known which contains Salsola Collina herb dry extract as activesubstance.

Use of this plant preparation based on Salsola Collina is equivalent tothe use of a combination of chemical substances because, as stated inthe patent description, the active complex contains flavonoids,polysaccharides, carotinoids, sterines, saponins, lipids, amino acidsand trace elements. As with above analogue, the variety like this isequivalent to polyprogmasy and will interfere with correctinterpretation of patient's response to treatment. Furthermore, thecontent of active substance in the drug varies from 0.25 to 99.9% whichis indicative of a low level of the drug standardization.

Other cholesterol-lowering drugs based on chemical preparations areknown. Of particular interest are such drugs as nicotinic acid and itsderivatives, bile acids sequestrants, fibric acid derivatives(fibrates), probucol (see Okorokov, A. N. Treatment of InternalDiseases/Practical Guide. Minsk: Vysheshaya Shkola, Vitebsk:Belmedkniga, 1996, vol. 3, book 1, pp. 19-41).

However, treatment with nicotinic acid is associated with a number ofside effects, such as liver function impairment, exacerbation of chronicgastritis, gastric ulcer, and also can cause hyperglycemia, skinhyperemia and an elevation of blood levels of uric acid.

Therapy with sequestrants is associated with such side effects asnausea, flatulence, constipation and diarrhea. The treatment can alsolead to elevation of triglycerides levels and impairment of digestion offat-soluble vitamins (A, D, K) and folic acid.

Side effects of the use of fibrates (e.g. bezafibrate, fenophibrate,gemfibrozil) are muscle affection, an increase of lithiasis in biliarytracts, and a possibility to cause leucopenia, thrombocytopenia, andanemia.

Probucol favors ventricular arrythmias, dyspeptic disorders and canincrease liver dysfunction.

Lovastatin (synonyms: Mevacor, Lovacor, Medostatin, Recol, Rovacol), acholesterol-lowering drug, which regulates metabolic processes andcontains an active substance of a chemical origin and special-purposeexcipients, is the most closely related art drug to the claimed drug(see Vidal's Guide. Drugs in Russia. Moscow: AstraFarmServis, 2000, pp.B-364 to B-365).

The active substance of the related art drug belongs to statins and is[IS(I α(R*),3α,7β,8β(2S*,4S*),8αβ]1,2,3,7,8α,-Hexahydro-3,7-dimethyl-8[2-(tetrahydro)-4-hydroxy-6-oxo-2H-pyran-2-yl]-1-naphthalenyl2-methyl butanoate. The substance acts by inhibiting cholesterolbiosynthesis. In the body, lovastatin is metabolized to give β-oxyacidwhich is a competitive inhibitor of an enzyme3-hydroxy-3-methyl-glutaryl coenzyme A (HMG-CoA) reductase.

The related art drug also contains lactose, gelated starch,microcrystalline cellulose, butylated hydroxyanisol, indigotin (E132),and magnesium stearate as excipients.

A drawback of statins, including lovastatin, is that these drugs shouldbe taken for long. Furthermore, statins can affect liver, muscles,gastrointestinal tract, cause sleep disturbances, headaches. In repeateddrug administrations, the efficacy of the drug therapy can be reduced(tachyphylaxis) (see Cromwell W. C. Ziajka P. E. Development oftachyphylaxis among patients taking HMG CoA reductase inhibitors./ Am J.Cardiol 2000; 86: 1123-1127// The translation is published in CONSILIUMmedicum journal, 2001, vol. 3, no. 2. Translated by Elagin, R. I.Razvitie takhifilaksii u patsientov, poluchayushchikh ingibitory HMG-CoAreductasy). In addition, statins show xenobiotic properties.

BRIEF DESCRIPTION OF THE INVENTION

The goal of the present invention is to develop a physiologicallyacceptable cholesterol-lowering preparation causing no toxicity andtachyphylaxis.

The mentioned goal is achieved by providing the inventive cholesterolcontrol agent that includes an aqueous solution of formaldehyde with36.5-40% concentration as an active substance and an isotonic solutionof sodium chloride for injections with 0.85-0.95% concentration as aspecial-purpose additive (excipient) at the following correlation of theweight parts (units by weight):

aqueous solution of formaldehyde with 36.5-40% concentration - 2-6isotonic solution of sodium chloride for injections 994-998 with0.85-0.95% concentration

To the authors' knowledge, no data are available in patents andscientific-technical literature on any drugs lowering cholesterol levelsthrough increasing receptor affinity.

Formaldehyde is known to be a natural product of cellular metabolism(see Hunter B. K. et al. Formaldehyde metabolism by Escherichia coli.Carbon and solvent deuterium incorporation into glycerol,1,2-propanediol and 1,3-propanediol.// Biochemistry, 1985, vol. 24, no.15, pp. 4148-4155). Formaldehyde is produced as a result of metabolicactivities from alcohols and amino acids in eukaryotes and prokaryotesand found in cells either free or bound to other metabolites, primarily,to tetrahydrofolate and glutathione. Formaldehyde is also known to beused as immune-modulator (see RF Patent No. 2077882 according to IPCA61K31/115, published on Apr. 27, 1997).

The mechanism of regulation of blood cholesterol is based on the abilityof formaldehyde, discovered by the authors, to trigger a hypermutationmechanism, which leads to an increase of the affinity of receptorsregulating the cholesterol levels.

The instant authors have established experimentally that introduction offormaldehyde ‘in vitro’ leads to a decrease of the level of thetransforming growth factor, which provokes apoptosis or programmed celldeath. Apoptosis participates in pathogenesis of atherosclerosis ofheart coronary vessels (see, for example, Storozhakov G. I., Uteshev D.B. Apoptosis' role in the development of atherosclerosis, myocardialischemia and cardiac insufficiency/Cardiac insufficiency—2000.—vol.1.—No 4). In pathogenesis of atherosclerosis one of the importantaspects is impairment of endothelium function. Now endotheliumdysfunction is understood as (see, for example, Cherkashin D. V.Clinical significance and correction of endotheliumfunction—htt:/www.Cardiolog.ru) imbalance between mediators providingnormally optimal course of all endothelium depending processes,including also the transforming growth factor, relating to prosclerouscytokines (see, for example, Cooper M. E., Rumbler Y., Komers R. et.al.—Diabets.—1994.—No 43—P. 1221-1228).

At that not only abnormal cells of vascular walls but also of othertissues such as liver, thyroid, adrenals are exposed to apoptosis. This,in turn, results in functional recovery of the organs, includingreactivation of enzymes responsible for lipid breakdown, andnormalization of a physicochemical condition of blood proteins which,ultimately, leads to normalization of the blood cholesterol levels.

An aqueous solution of formaldehyde is a transparent colorless liquidwith a specific sharp odor, miscible with water and alcohol in allproportions.

Formaldehyde belongs to the class of aldehydes HCOH and is a colorlessgas with a sharp odor, having a molecular weight of 30.03, density (at20° C.) of 0.815, melting point of −92° C. and boiling point of −19.2°C. It is well soluble in water and alcohol. The substance is easilypolymerized to form paraformaldehyde in an aqueous medium orpolyoxymethylene in non-aqueous media (butane, hexane).

Isotonic solution of sodium chloride for injections is a colorlessliquid with saline taste. The solution is sterile and apyrogenic.

Sodium chloride is cubic crystals or white crystalline powder withsaline taste, odorless and soluble in water (1:3).

The claimed preparation (cholesterol control agent) is a transparentcolorless solution, that is odorless slightly saline in taste.

DETAIL DESCRIPTION OF PREFERRED EMBODIMENTS OF THE INVENTION

While the invention may be susceptible to embodiment in different forms,there are described in detail herein, specific embodiments of thepresent invention, with the understanding that the present disclosure isto be considered an exemplification of the principles of the invention,and is not intended to limit the invention to that as exemplifiedherein.

Inventive Preparation and Method Therefor.

The preparation is preferably made in the following way:

Take 2-6 weight parts of 36.5-40% medicinal solution of formaldehyde,add it to 998-994 (accordingly to make the total of 1000 weight parts)weight parts of sterile 0.85-0.95% solution of sodium chloride forinjections to receive 0.07-0.24% solution of formaldehyde. Thepreparation should be stored in a dark place at temperature of 15-35° C.

Example 1

Take 0.2 ml of 37% medicinal solution of formaldehyde, add it to 99.8 mlof sterile 0.9% (or 0.95%) isotonic solution of sodium chloride. The soobtained solution mixture is stirred thoroughly. The final concentrationof formaldehyde in the received preparation will make 0.074 mass %.

Example 2

Take 0.6 ml of 37% medicinal solution of formaldehyde, add it to 99.4 mlof sterile 0.9% (or 0.95%) isotonic solution of sodium chloride. The soobtained solution mixture is stirred thoroughly. The final concentrationof formaldehyde in the received preparation will make 0.222 mass %.

Example 3

Take 0.6 ml of 40% medicinal solution of formaldehyde. Make thepreparation according to the method described in example 2. The finalconcentration of formaldehyde will make 0.24 mass %.

Example 4

Take 3 ml of 40% medicinal solution of formaldehyde, add it to 997 ml ofsterile 0.95% isotonic solution of sodium chloride. The so obtainedsolution mixture is stirred thoroughly. The final concentration offormaldehyde in the received preparation will make 0.12 mass %.

Experiments on Toxic Effect

To prove the absence of any toxic effect of the claimed preparation,experiments on mice were performed.

The experimental study was conducted on 56 outbreed ICR male and femalemice weighing 18 to 24 g. The experimental animals were kept in astandard living and care environment of a vivarium on steady diet andprovided with unlimited access to water and feed.

Animal observations were carried out during 14 days post administrationof the preparation. During the follow-up period, general condition,motor activity, behavior, reaction to stimuli and reflexes wereassessed. When required by the protocol of experiments, animals wereweighted and consumptions of food and water were determined. Theanimals, died during the experiments, were dissected and an assessmentof changes was made and organ weight ratios were determined (brain,liver, heart, spleen, kidneys, adrenals, testicles, ovaries). Thesurvived animals were crucified at the end of the follow-up period bydislocation of the cervical spine and dissected. A postmortemexamination was conducted to assess macroscopic changes of theinternals, if any.

Toxicity was assessed by the Litchield and Wilcoxon method of probitanalysis using S. Rot's nomograms.

The preparation containing 0.2 mL of 37% formaldehyde solution and 99.8mL of 0.9% sodium chloride solution was administered onceintramuscularly at doses of 5, 25 and 50 mL/kg body weight. During thefollow-up period, test animals were weighted trice (1, 7 and 14 dayspost dose administration).

The study did not reveal any changes in general condition and behaviorof the test animals after injection of the preparation at a dose of 5.0mL/kg. There was no case of animal death during the follow-up period.Weight gains were within the normal range. No difference was found ingeneral condition and behavior between male and female animals.

In the 25 mL/kg group, the picture was close to that described above.General condition, behavior, food and water consumptions, weight gainswere within normal ranges both in male and female animal subgroups.

In the 50 mL/kg group, animals showed a short-term (lasting up to 1 min)slight agitation, but after that the condition of the animals in thisgroup did not differ significantly from that in other experimentalgroups.

At the end of the follow-up period (14 days post dose administration),test animals were crucified by dislocation of the cervical spine,dissected and subjected to macroscopic examination. After that, theinternals were removed, weighted and organ weight ratios weredetermined. The post mortem study showed that organ weight ratios didnot differ significantly between experimental groups and were withinnormal ranges. No local reactions (infiltrate, hyperemia, hair loss,etc.) were observed.

In another study, the preparation was administered at a concentration ashigh as 10 times the strength of drug product, i.e. containing 2 mL of37% formaldehyde solution and 98 mL of 0.9% sodium chloride solution.

The preparation was administered i.m. to ICR mice (male and female) atdoses of 6, 12.5 and 25 mL/kg.

During the first 5-6 hours post dose administration, test animals showeda moderate depression of excitability and reactivity. There was agradual acceleration of breathing and heart beating. However, no case ofanimal death was registered during the first 24 h post administration ofthe preparation. Only one mouse died from respiratory arrest on day 4post administration of the dose of 25 mL/kg.

Post dose administration, all animals developed a local reaction asmuscular tissue induration. At the end of the experiment, hair loss andformation of dense infiltrate were seen at the site of injection.

The study showed that the initial toxic dose did not have anysignificant influence on weight gain of the test animals. Variations ofthe values of weight gain in male and female groups were within limitsof tolerance.

At the end of the experiment, test animals were crucified and subjectedto macroscopic examination, the internals were removed, weighted andorgan weight ratios were calculated.

The study showed that in test animals, both male and female, whoreceived the preparation at a dose as high as 10 times therapeutic dose,organ weight ratios for brain, heart, liver, spleen, kidney, adrenals,testicles (ovaries) at the end of study period did not differsignificantly from respective normal values in general population.

Thus, the results obtained suggest a low toxicity of the appliedpreparation.

Inventive Method of Treatment

The inventive method of treatment in preferred embodiments consists inthat the inventive preparation is administered given as intramuscularinjections at a predetermined dose, preferably of 5 mL at predeterminedtime intervals, preferably: 7, 21, 30, and 60-day intervals.

To measure the cholesterol levels in patients, venous blood samples weretaken from the ulnar vein in the morning hours.

The clinical study of the preparation was conducted at RepublicanInfectious Hospital of the Republic of Belarus (Minsk) in compliancewith principles of Declaration of Helsinki, requirements ofinternational ethic standards and scientific standards of quality anddesign of studies involving human subjects, Public Health Law of theRepublic of Belarus and Order of the Ministry of Health of the Republicof Belarus No. 254 dated Aug. 18, 2000 On Conducting Clinical Studies ofDrugs.

Efficacy of the treatment was determined 7, 28, 35 and 65 days post doseadministration. Patients with cholesterol levels up to 5.2 mM/L areconsidered normal (see Okorokov, A. N. Treatment of InternalDiseases/Practical Guide. Minsk: Vysheshaya Shkola, Vitebsk:Belmedkniga, 1996, vol. 3, book 1, pp. 19-41), from 5.2 to 6.2 mM/Lmoderately and higher than 6.2 mM/L heavily hypercholesterinemic.

The results of cholesterol level correction are given in Table 1.

TABLE 1 Blood serum cholesterol levels, mM/L 7 days post dose 28 dayspost dose 35 days post dose 65 days post dose Baseline administrationadministration administration administration (M ± m) (M ± m) (M ± m) (M± m) (M ± m) 4.35 ± 0.26 4.67 ± 0.45 5.31 ± 0.46 3.58 ± 0.15 3.17 ± 0.18Treatment efficacy (n = 20) P < 0.05 for differences between days 35 and65 and baseline.

Data presented in Tables 2 and 3 are demonstrative of the efficacy oftreatment in patients with moderate and heavy hypercholesterinemia.

TABLE 2 Results of treatment of moderately hypercholesterinemic patientsBlood serum cholesterol levels, mM/L 7 days post dose 28 days post dose35 days post dose 65 days post dose Patient Baseline administrationadministration administration administration K. 5.4 8.5 8.5 4.6 3.6 P.5.7 4.3 3.0 2.8 4.6

TABLE 3 Results of treatment of heavily hypercholesterinemic patientsBlood serum cholesterol levels, mM/L 7 days 28 days 35 days 65 daysPatient Baseline post dose post dose post dose post dose V. 6.8 7.3 4.63.08 2.8 O. 6.8 3.3 4.6 3.2 1.96

Thus, the claimed preparation lowers blood cholesterol levels withoutcausing toxicity and tachyphylaxis.

1. A preparation, possessing cholesterol-lowering action, consistingessentially of: an active substance in the form of an aqueous solutionof formaldehyde with a concentration of 36.5-40%, said active substanceconstitutes from 2 to 6 weight units; and an additive in the form of anisotonic solution of sodium chloride with a concentration of 0.85-0.95%,said additive constitutes from 998 to 994 weight units accordingly, tomake the total of 1000 weight units.
 2. A method for obtaining apreparation comprising the steps of: providing an aqueous solution offormaldehyde with a concentration of 36.5-40% in the amount of from 2 to6 weight units; providing an isotonic solution of sodium chloride with aconcentration of 0.85-0.95% in the amount of from 998 to 994 weightunits; and adding said aqueous solution into said isotonic solutionthereby obtaining said preparation.
 3. The method of claim 2, furthercomprising the step of storing said preparation in a dark place attemperature of 15-35° C.
 4. A method for treatment of patients forlowering their cholesterol levels comprising: administering thepreparation of claim 1 to the patients as intramuscular injections witha predetermined dose at a predetermined time interval.
 5. The method ofclaim 4, wherein said predetermined dose constitutes 5 mL.
 6. The methodof claim 4, wherein said predetermined time interval is selected fromthe group consisting of 7, 21, 30, and 60-day intervals.